Progress 05/21/15 to 09/30/17
Outputs
Target Audience:Scientific researchers in forest nutrient cycling and mycorrhizal ecologists. Changes/Problems:We report results pertaining to sap flow, nutrient uptake, and mycorrhizal fungi. The proposals to fund related work as part of a larger project, led by Matt Vadeboncoeur at University of New Hampshire and Mark Green at Plymouth State University were not successful. We did succeed at characterizing how nutrient availability affects water use, nutrient uptake, and mycorrhizal community composition. But we do not have data on how mycorrhizae affect water uptake, which was one of the original goals of the project. We also do not have evidence that transpiration is modified by nutrient addition to improve uptake of limiting nutrients. What opportunities for training and professional development has the project provided?The following students have been supported on this award: 2017:Alexandrea Rice, Daniel Hong and Gretchen Lasser. Claudia Victoroffis supporting this project but not being financially compensated. This project represents an important part of work toward Alexandrea Rice and Claudia Victoroff's master's theses. Daniel Hong and Gretchen Lasser provided support for field activities. 2016: Jerome Barner was supported on this award and completed his master's thesis, Ectomycorrhizal fungi contribution to nutrient cycling of nitrogen, phosphorus, and calcium in northern hardwood forests. The following undergraduate students worked in Tom Horton's molecular lab during the academic year processing samples: Austin Frewert, Marrissa Lanzatella and Jalina Pannafino. 2015: Brigid Farrell (graduate student) and Isaac Jo, Jesse Smith, Matthew Hayden (undergraduates) worked on the sap flow project. How have the results been disseminated to communities of interest?Alexandrea Rice presented her work at the following scientific meetings: • 54th Annual Hubbard Brook Cooperators' Meeting, North Woodstock, NH, July 13, 2017 • Rochester Academy of Sciences Fall Scientific Paper Session, St. John Fisher College, November 11, 2017 • 2017 Forest Ecosystem Monitoring Cooperative Conference, University of Vermont, December 15, 2017 Claudia Victoroff presented her work at the following scientific meetings: • 54th Annual Hubbard Brook Cooperators' Meeting, July 13, 2017 • IMC11 (International Mycological Congress) conference, San Juan, Puerto Rico, to be held in July 16-21, 2018 (expected) Publication in preparation: Alexandrea Rice, Ruth Yanai and Mariann Johnston. Nutrient availability affects sap flux in a northern hardwood forest. Forest Ecology and Management (intended publication). This abstract was submitted to the IMC11 (International Mycological Congress) conference, San Juan, Puerto Rico, to be held in July 16-21, 2018. Thomas R. Horton1, L. Jamie Lamit1, Peter Kennedy2, Ruth Yanai3 Using MiSeq on DNA extracted from in-growth bags to observe ectomycorrhizal fungi with N, P and N+P additions in mature forest plots in Bartlett Experimental Forest, New Hampshire. 1Department of Environmental and Forest Biology, SUNY-ESF, 1 Forestry Drive, Syracuse, NY 13210 2College of Biological Sciences, University of MN, 1479 Gortner Avenue, St. Paul, MN 55108 3Forest and Natural Resources Management, SUNY-ESF, 1 Forestry Drive, Syracuse, NY 13210 Forests growing on young, recently glaciated soils are predicted to be N limited in early succession and then become more P limited as they age. Experimental tests of N vs. P limitation in such forest systems are few, and those few have been short-term with very high rates of fertilization. Further, while there has been work on the response of mycorrhizal fungi to N additions, little work been conducted with P. We investigated ectomycorrhizal (EM) fungi in mature forest soils exposed to nutrient additions in the Bartlett Experimental Forest, NH. N (30 kg N/ha/yr as NH4NO3), P (10 kg P/ha/yr as NaH2PO4) and N+P were added annually starting in 2011 in an experiment to investigate multiple element limitation in northern hardwood ecosystems. We used hyphal ingrowth bags to study the response of soil fungi to nutrient additions in three stands. After two growing seasons, the bags were harvested and DNA extracted from the soil. We generated community profiles from these extracts based on the fungal ITS1 region, using the Illumina MiSeq platform. Sequence data were processed using the BBMap package, VSearch 2.5.1 and Qiime 1.9, while FUNGuild was used to identify EMF taxa. As expected, N additions reduced the richness of OTUs identified as EM taxa. P additions did not change the richness of EM taxa compared to the controls, and plots with N+P additions had intermediate levels of richness. Treatment effects were observed in the assemblages of dominant genera, dominance being measured as the total number of sequences recovered. In control plots the three most dominant genera were Tomentella, Tuber and Tomentellopsis. In N plots the three most dominant genera were Sebacina, Tuber and Tomentella. In P plots the three most dominant genera were Genea, Pseudotomentella and Pachyphleous. In N+P plots the three most dominant genera were Paxillus, Tomentella and an unidentified member of the Ceratobasidiaceae. The majority of sequences recovered for a genus in each nutrient treatment were assigned to one OTU in the genus, the exception being Tomentella in control plots with three high abundance OTUs. These data provide a picture of EM fungi in soils as active mycelial networks under the various nutrient treatments. Further, these are some of the first such data from P addition plots and identify some EM taxa that may be functioning in forest P cycling. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Transpiration is the movement of water and nutrients from the soil to the atmosphere via xylem transportation. This is controlled through photosynthesis and several environmental variables such as the amount of sunlight present and humidity and has a direct effect on the water cycle in an ecosystem. In today's climate models, transpiration plays a large role in determining forest water budgets, however, current water budgets fail to incorporate the role of nutrient availability and its effect on nutrient uptake and sap flux rates. We took advantage of a long-term Multiple Element Limitation in Northern Hardwood Ecosystems (MELNHE) study, established in 2011, that incorporated three locations, Bartlett Experimental Forest, Jeffers Brook Forest, and Hubbard Brook Experimental Forest, located in the White Mountain National Forest, New Hampshire, USA (44?2'60" N, 71?16'48" W). A total of 13 stands represented three successional age classes: young, mid-aged, or mature. Each of these stands included 0.25- ha plots treated with 30 kg ha-1yr-1 of nitrogen as NH4NO3, 10 kg ha-1yr-1 of phosphorus as NaH2PO3, both N and P combined, or was a non-fertilized control. Seven stands also contained a calcium addition plot that received a one-time application of wollastonite (CaSiO2) of 1150 kg ha-1 of calcium in October 2011. Our results are not conclusive, but our experiments suggest that sap flow in sugar maple may be sensitive to calcium, if notnitrogen or phosphorus. We found differences in fungal communities in the different nutrient treatments. More research is warranted on whether mycorrhizal fungal associations may contribute to variation in water use as well as on the importance of including nutrients in predicting transpiration and water use. What was accomplished under these goals? 1. Sap Flux Sap flux in white birch was measured during July and August 2015 at Bartlett Experimental Forest, in a mid-aged stand, C6. Using the Granier (1987) thermal dissipation method, we measured sap flow in 23 trees distributed among the 5 treatment plots (control, N, P, N+P, Ca). In July and August 2017, sap flux in sugar maple was measured in a mature stand, C8, also at Bartlett. We instrumented 25 trees among the 5 treatment plots (control, N, P, N+P, Ca). Data were converted from temperature differential to sap flux using the program BaseLiner. Analysis of variance was used to test for effects of nutrient treatments on sap flux. In white birch, we found no significant effect on sap flux with the addition of N, P, or N+P (p=0.19). The effect of Ca addition was also not significant (p=0.21). In sugar maple, similarly, the addition of N, P, and N+P had no significant effect on sap flux (p=0.29). There was however an increase in sap flux with Ca additions (p=0.06). This increase in sap flux with the addition of Ca was also observed in 2014 in Jeffers Brook and Hubbard Brook Experimental Forest, with 54 trees total of American beech, sugar maple, and yellow birch, in preliminary measurements conducted prior to the funding of this study. This finding is exciting as it helps to explain an earlier observation of a reduction in stream flow following a watershed-level addition of Ca at Hubbard Brook. 2. Nutrient uptake Nutrient uptake was measured in red maple and in yellow birch in a young stand at Bartlett (C2). Intact roots branches were excavated by hand from the organic and upper mineral soil horizons, rinsed with DI water to remove soil, and exposed to 27 mL of nutrient solution while still attached to the tree. The tubes containing the solution were covered to prevent evaporation and and aerated to supply the roots with oxygen. Nutrient concentrations of 1, 5, and 10 times the soil solution concentration were applied sequentially to each root. After 2 hours, the solution was filtered and analyzed by ICP-OES for Al, Ca, Mg, K, P, and S. Ammonium and nitrate were analyzed using a continuous flow analyzer. At the end of the experiment, roots were removed for wet weight measurement and imaging for root length. Uptake of most elements were not significantly affected by nutrient treatments, except that Ca efflux from roots was lowest in the NP treatment plot (p=0.04). This technique might hold promise for additional study, now that data processing errors by a previous student have been corrected. 3. Fungal community In-growth sand bags were first used to bait for ectomycorrhizal fungi (EMF) by Nilsson and Wallander (2003). Jerome Barner and Tom Horton installed in-growth sand bags (15 cm2 pouches with 0.44 µm nylon mesh; Plastok Associates Limited) in the MELNHE plots in July of 2016. Bags were filled with autoclaved play sand, inserted into the soil vertically starting just below the litter layer. Bags were installed in plots A1-4, B1-4, C1-4 and D1-4. Bags were installed in N, P and N+P addition plots, and the unamended control. The bags were harvested in November 2016 (i.e., growing season for most EMF in this region). The DNA was extracted from the sand using a MoBio PowerSoil DNA Isolation Kit. We generated community profiles from these extracts based on the fungal ITS1 region, using the Illumina MiSeq platform. We used modified ITS-2 primers with unique barcodes to track individual samples in a MiSeq run (Smith and Peay 2014, Kennedy pers. comm.). Sequence data were processed using the BBMap package, VSearch 2.5.1 and Qiime 1.9, while FUNGuild was used to identify EMF taxa. Tom Horton prepared the samples and did the MiSeq run under the guidance of Karen Gentile in Frank Middleton's lab at the SUNYMAC sequencing facility (UPSTATE). We processed the resulting sequence data under the guidance of Jamie Lamit, a postdoc in the departments of EFB at SUNY-ESF and Biology at Syracuse University. We used the BBMap package, VSearch 2.5.1 and Qiime 1.9, and FUNGuild to clean up the data and identify taxa of EMF. The number of DNA sequences retrieved in the run was impressively high, and we used a pipeline to filter out noise and chimeric sequences. We used a conservative approach for identifying Operational Taxonomic Units (OTUs), obtaining just over 1300 high quality OTUs. Treatment effects were observed in the assemblages of dominant genera, dominance being described by the three OTUs with the highest number of sequences recovered in a sample. In control plots the dominant genera were Tomentella, Tuber and Tomentellopsis. In N plots the dominant genera were Sebacina, Tuber and Tomentella. In P plots the dominant genera were Genea, Pseudotomentella and Pachyphleous. In N+P plots the dominant genera were Paxillus, Tomentella and an unidentified member of the Ceratobasidiaceae. The majority of sequences recovered for a genus in each nutrient treatment were assigned to one OTU in the genus, the exception being Tomentella in control plots with three high abundance OTUs. These data provide a picture of EMF in soils as active mycelial networks under the various nutrient treatments. Further, these are some of the first such data from P addition plots and identify taxa of EMF that may be functioning in forest P cycling. We will continue to analyze the data using EstimateS (Cowell 2006) to calculate various diversity measures using OTUs for comparison between tree genotypes (species richness, Simpson's diversity, Chao 1 and Chao 2, Jackknife estimator, etc.). Differences in fungal diversity measures among tree genotypes will be determined by analysis of variance (ANOVA) at α = 0.05 followed by a Tukey test.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Scientific researchers in forest nutrient cycling and mycorrhizal ecologists. Changes/Problems:We are focusing our efforts on measurements of sap flow rather than nutrient uptake as measurement of nutrient uptake has proven to be problematic. What opportunities for training and professional development has the project provided?Jerome Barner, a graduate student, has been supported on this grant. How have the results been disseminated to communities of interest?Presented at the Annual Hubbard Brook Cooperators meeting, July 2016. What do you plan to do during the next reporting period to accomplish the goals?We will publish the results to date and continue collecting more data in support of future grant proposals and publications.
Impacts
What was accomplished under these goals?
In 2014, transpiration flux rates (g/m2/sec) for sugar maple, yellow birchand American beechwere tested on control plots and plots that received a single 2011 treatment of wollastonite (CaSiO3) at three sites: Bartlett C8, Hubbard Brook Old, and Jeffers Brook Old. Results showed that for sugar maple and yellow birch, site was significant (p=0.01), with Bartlett showing higher transpiration rates than the other two sites. While treatment alone was not significant, a three-way interaction was detected between site, treatment and time (p=0.03). Graphical representation of this interaction suggests that theCaSiO3 treatment generally resulted in a more rapid rise in transpiration rates earlier in the day, and a higher peak rate of transpiration, compared to the control plots.Beech was analyzed separately, but demonstrated a similar pattern between the two treatment plots. A species effect between sugar maple and yellow birch was also detected (p=0.0p) with yellow birch showing higher transpiration rates than sugar maple, and a species by time interaction (p=0.001), with yellow birch transpiration rates rising more rapidly and peaking at a higher rate compared to sugar maple.In 2015, white birch was tested at a single site (Bartlett C6) on five treatments: Control, N, P, N+P (all annually recurring since 2011), and CaSiO3(single 2011 application). While treatment alone was not significant, the time by treatment interaction was (p=0.01), with the P treatment showing significantly lower transpiration rates than the other four treatments. Interestingly, transpiration rates on white birch in 2015 were approximately double the rates of the species tested in 2014, with the exception of the 2015 P plots, which approximated 2014 transpiration rates. Whether this was an effect of year or of species would require further field testing. In all cases, the explanatory effects of weather and time of day were important in being able to detect the effects of treatment, species and site on transpiration rates.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Barner J.C., T.R. Horton and R.D. Yanai. Belowground carbon allocation to ectomycorrhizal fungi associated with Fagus grandifolia in response to N, P, and Ca additions in northern hardwood forests. 8th International Conference on Mycorrhiza, August 3-8, 2015, Flagstaff AZ.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Johnston, M, S. Harrison, A. Wild and B. Farrell. Sap flow: Calcium, and what about N and P? Hubbard Brook Annual Meeting, July 13, 2016, W. Thonton, NH.
- Type:
Theses/Dissertations
Status:
Under Review
Year Published:
2016
Citation:
Barner J.C. Ectomycorrhizal fungi contribution to nutrient cycling of nitrogen, phosphorus, and calcium in northern hardwood forests. MS Thesis. State University of New York College of Environmental Science and Forestry.
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